Retinoic acid receptor antagonist inhibits CD38 antigen expression on human hematopoietic cells in vitro

The CD34+ CD38- subset of human hematopoietic stem cells are crucial for long-term ex-vivo expansion; conditions that decreased this specific sub-population reduced the self-renewal capacity and shortened the duration of the proliferative phase of the culture. Retinoids, such as all-trans retinoic acid (ATRA), have been shown to induce CD38 expression. ATRA present in serum may be responsible for the high CD38 of cells grown in serum-containing medium. In the present study we analyzed the effects of AGN 194310, a retinoic acid receptor pan-antagonist, on CD38 expression of human hematopoietic cells. Normal cells (cord blood derived CD34+ cells) and abnormal cells (myeloid leukemic lines) were studied when grown in either serum-containing or serum-free media. The results showed that both serum and ATRA enhanced differentiation and, thereby, reduced the proportion of CD34+ CD38- cells and total CD34+ cell expansion. AGN reversed these effects of serum and ATRA: it delayed differentiation and increased CD34+ CD38- cells. These results suggest that physiological ATRA levels in serum may prevent efficient cell expansion. AGN, by neutralizing ATRA, improves cell expansion in serum-containing cultures, thus making AGN a useful agent for ex vivo expansion of stem cells and other specific sub-populations for research and clinical use.     

Retinoic acid induction of CD38 antigen expression on normal and leukemic human myeloid cells: relationship with cell differentiation

Differentiation in the hematopoietic system involves, among other changes, altered expression of antigens, including the CD34 and CD38 surface antigens. In normal hematopoiesis, the most immature stem cells have the CD34+CD34 -phenotype. In acute myeloid leukemia (AML), although blasts from most patients are CD38+, some are CD38 -. AML blasts are blocked at early stages of differentiation; in some leukemic cells this block can be overcome by a variety of agents, including retinoids, that induce maturation into macrophages and granulocytes both in vitro and in vivo . Retinoids can also induce CD38 expression. In the present study, we investigated the relationship between induction of CD38 expression and induction of myeloid differentiation by retinoic acid (RA) in normal and leukemic human hematopoietic cells. In the promyelocytic (PML) CD34 -cell lines, HL60 and CB-1, as well as in normal CD34+CD34 -hematopietic progenitor cells RA induced both CD38 expression as well as morphological and functional myeloid differentiation that resulted in loss of self-renewal. In contrast, in the myeloblastic CD34+ leukemic cell lines, ML-1 and KG-1a, as well as in primary cultures of cells derived from CD34+-AML (M 0 and M 1 ) patients, RA caused an increase in CD38+ that was not associated with significant differentiation. Yet, long exposure of ML-1, but not KG-1, cells to RA resulted in loss of self-renewal. The results suggest that while in normal hematopoietic cells and in PML CD34 -cells induction of CD38 antigen expression by RA results in terminal differentiation along the myeloid lineage, in early myeloblastic leukemic CD34+ cells, induction of CD38 and differentiation are not functionally related. Since, several lines of evidence suggest that the CD38 -cells are the targets of leukemic transformation, transition of these cells into CD38+ phenotype by RA or other drugs may have therapeutic effect, either alone or in conjunction with cytotoxic drugs, regardless the ability of the cells to undergo differentiation.     

Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities

The destruction of cells capable of initiating and maintaining leukemia challenges the treatment of human acute myeloid leukemia. Recently, CD34+/CD38- leukemia progenitors have been defined as new leukemia-initiating cells less mature than colony-forming cells. Here we show that CD34+/CD38- leukemia precursors have reduced in vitro sensitivity to daunorubicin, a major drug used in leukemia treatment, in comparison with the CD34+/CD38+ counterpart, and increased expression of multidrug resistance genes (mrp/lrp). These precursors show lower expression of Fas/Fas-L and Fas-induced apoptosis than CD34+/CD38+ blasts. Moreover, the CD34+/CD38- leukemic subpopulation induces a weaker mixed leukocyte reaction of responding T-lymphocytes than the CD34+/CD38+ leukemic counterpart, either in a MHC-unmatched or MHC-matched settings. This weaker immunogenicity could be linked to lower expression on CD34+/CD38- leukemia precursors of major immune response molecules (MHC-DR, LFA-3, B7-1, or B7-2) than CD34+/CD38+ leukemic cells. Nonetheless, the susceptibility of the immature CD38- precursors to cytotoxicity was not different from the sensitivity of the CD38+ counterpart. Finally, CD34+/CD38- leukemia precursors, in contrast with CD38+ precursors, failed, under appropriate conditions, to differentiate into dendritic cells, a central step for antigen recognition. This is to our knowledge the first demonstration that the very immature phenotype of CD34+/CD38- leukemic progenitors confers both chemotherapy resistance and decreased capacities to induce an immune response. Because the susceptibility of the immature leukemia cells as cytotoxic targets is maintained, our data underline the importance of improving the initial steps of leukemia recognition, more particularly by defining optimal conditions of dendritic cell transformation of the very immature hematopoietic precursors.     

Severe functional alterations in vitro in CD34(+) cell subpopulations from patients with chronic myeloid leukemia

Chronic myeloid leukemia (CML) arises from the malignant transformation of a hematopoietic stem cell (HSC) that gives rise to functionally defective progeny, including primitive and relatively mature progenitor cells (HPC). Both HSC and HPC are comprised within the population of CD34(+) cells, normally present in bone marrow (BM). In the present study, we have separated two different subpopulations of CD34(+) cells from CML marrow: Population I, enriched for CD34(+) Lin(-) cells; and Population II, enriched for CD34(+) CD36(-) CD38(-) CD45RA(-) Lin(-) cells, and assessed their progenitor cell content as well as their capacity to proliferate and expand in response to a combination of hematopoietic cytokines in serum- and stroma-free long-term liquid cultures. The absolute cell numbers recovered in Population I from normal and CML samples were similar; in contrast, we found that Population II from CML was amplified four-fold, as compared to normal. In spite of this latter observation, no significant differences were observed in terms of the absolute number of CFC when comparing Populations I and II from CML patients and normal subjects. Interestingly, the proliferation and expansion potentials of CML cells were clearly deficient as compared to their normal counterparts. Indeed, in cultures of Population I cells the maximum fold increase in total and progenitor cell numbers corresponded to 30 and 8%, respectively, of those observed in cultures of normal marrow-derived Population I cells. Such functional deficiencies were even more evident in Population II cells in which the maximum fold increase in total and progenitor cell numbers corresponded to 3 and 0.5%, respectively, of the levels found in cultures of Population II cells from normal marrow. The present study demonstrates that bone marrow-derived CD34(+) cells from CML patients possess functional abnormalities, clearly evident in the in vitro system used by us. Among the two CML subpopulations studied here, the more immature one (Population II; enriched for CD34(+) CD36(-) CD38(-) CD45RA(-) Lin(-) cells) was the one that showed the most severe abnormalities, as compared to its relatively more mature counterpart (Population I; enriched for CD34(+) Lin(-) cells).     

Stem cell factor and chronic myeloid leukemia CD34+ cells

Normal hematopoiesis is a tightly regulated process involving a balance between signals that stimulate and those that inhibit the proliferation and differentiation of hematopoietic progenitors. In chronic myeloid leukemia (CML) there is a perturbation of these controlling elements, resulting in overgrowth of leukemic cells in the bone marrow and spleen. In part, the proliferation of CML CD34+ cells may result from an abnormal response to the cytokine Stem Cell Factor (SCF). SCF induced proliferation and adhesion to the extracellular matrix via fibronectin are not coupled in CML as they are in normal cells and this may contribute to the accumulation of leukemic progenitors. We have previously shown that CD34+ CML cells and the more primitive CD34+ CD38- CML cells do not require the addition of synergistic cytokines to cultures, but are capable of proliferation in SCF alone, and that leukemic CFU-GM are selectively supported in these cultures. In the presence of other cytokines the response of CML cells to SCF is no greater than that of cells from normal donors, suggesting that the leukemic cells are not more sensitive to SCF, but that accessory pathways are already activated in these cells. Cells from patients with myeloproliferative disorders show variable proliferative response to SCF as the sole mitogenic stimulus, suggesting that expression of bcr-abl is essential for proliferation in this cytokine. Further studies to identify the key determinants of the abnormal response to SCF in CML may lead to a better understanding of the proliferative abnormality that underlies CML.     

Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34+CD38- cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells.

Primitive hematopoietic progenitor cells such as severe combined immunodeficiency- repopulating cells and long-term culture-initiating cells are enriched in CD34+CD38- cells derived from various stem cell sources. In this study, to elucidate the features of such primitive cells at the molecular level, we tried to isolate genes that were preferentially expressed in umbilical cord blood (CB)-derived CD34+CD38- cells by subtractive hybridization. The gene for VPAC1 receptor, a receptor for the neuropeptide vasoactive intestinal peptide (VIP), was thereby isolated and it was shown that this gene was expressed in both CD34+CD38- and CD34+CD38+ CB cells and that the expression levels were higher in CD34+CD38- CB cells. Next, we assessed the effects of VIP on the proliferation of CD34+ CB cells using in vitro culture systems. In serum-free single-cell suspension culture, VIP enhanced clonal growth of CD34+ CB cells in synergy with FLT3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO). In serum-free clonogenic assays, VIP promoted myeloid (colony-forming unit-granulocyte/macrophage (CFU-GM)) and mixed (CFU-Mix) colony formations. Furthermore, in Dexter-type long-term cultures, VIP increased colony-forming cells at week 5 of culture. These results suggest that VIP functions as a growth-promoting factor of CB-derived hematopoetic progenitor cells.     

CD34high+ CD38(low/-) cells generated in a xenogenic coculture system are capable of both long-term hematopoiesis and multiple differentiation.

CD34+ cells isolated from human umbilical cord blood (HUCB) are thought to have potential in clinical applications such as transplantation and gene therapy. Recently, we developed a xenogenic coculture system involving HUCB-CD34+ cells and murine bone marrow stromal cells, HESS-5 cells, in combination with human interleukin-3 and stem cell factor. Under these xenogenic coculture conditions, the numbers of CD34high+ cells and primitive progenitor cells, such as CD34high+ CD38(low/-) cells and high proliferative potential colony-forming cells (HPP-CFCs), increased dramatically by a factor of 102.1, 66.5 and 104.9, respectively. In the present study, we used a secondary culture of B progenitor cells and long-term culture (LTC)-initiating cells to characterize and compare the progenitor capability of re-isolated CD34high+ CD38(low/-) cells, which have been identified as one of the most primitive progenitor cells, with that of freshly isolated CD34high+ CD38(low/-) cells. Compared with freshly isolated CD34high+ CD38(low/-) cells, the re-isolated CD34high+ CD38(low/-) cells were equally as capable of proliferating and differentiating into myeloid and B progenitor cells. No significant differences were observed in the frequency of LTC-initiating cells in the re-isolated CD34high+ CD38(low/-) cells compared with that in freshly isolated CD34high+ CD38(low/-) cells. Furthermore, the re-isolated CD34high+ CD38(low/-) cells were capable of long-term reconstitution and multiple differentiation in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). The results demonstrate that this xenogenic coculture system can be used for successful in vitro expansion of HUCB-progenitor cells that possess the capability for both long-term hematopoiesis as well as multipotent differentiation into myeloid and lymphoid cells both in vivo and in vitro.     

Expression of HOX genes, HOX cofactors, and MLL in phenotypically and functionally defined subpopulations of leukemic and normal human hematopoietic cells.

To explore the possibility that deregulated HOX gene expression might commonly occur during leukemic hematopoiesis, we compared the relative levels of expression of these and related genes in phenotypically and functionally defined subpopulations of AML blasts and normal hematopoietic cells. Initially, a semi-quantitative RT-PCR technique was used to amplify total cDNA from total leukemic blast cell populations from 20 AML patients and light density cells from four normal bone marrows. Expression of HOX genes (A9, A10, B3 and B4), MEIS1 and MLL was easily detected in the majority of AML samples with the exception of two samples from patients with AML subtype M3 (which expressed only MLL). Low levels of HOXA9 and A10 but not B3 or B4 were seen in normal marrow while MLL was easily detected. PBX1a was difficult to detect in any AML sample but was seen in three of four normal marrows. Cells from nine AML patients and five normal bone marrows were FACS-sorted into CD34+CD38-, CD34+CD38+ and CD34-subpopulations, analyzed for their functional properties in long-term culture (LTC) and colony assays, and for gene expression using RT-PCR. 93 +/- 14% of AML LTC-initiating cells, 92 +/- 14% AML colony-forming cells, and >99% of normal LTC-IC and CFC were CD34+. The relative level of expression of the four HOX genes in amplified cDNA from CD34- as compared to CD34+CD38- normal cells was reduced >10-fold. However, in AML samples this down-regulation in HOX expression in CD34- as compared to CD34+CD38- cells was not seen (P < 0.05 for comparison between AML and normal). A similar difference between normal and AML subpopulations was seen when the relative levels of expression of MEIS1, and to a lesser extent MLL, were compared in CD34+ and CD34- cells (P < 0.05). In contrast, while some evidence of down-regulation of PBX1a was found in comparing CD34- to CD34+ normal cells it was difficult to detect expression of this gene in any subpopulation from most AML samples. Thus, the down-regulation of HOX, MEIS1 and to some extent MLL which occurs with normal hematopoietic differentiation is not seen in AML cells with similar functional and phenotypic properties.     

Different subsets of primary chronic myeloid leukemia stem cells engraft immunodeficient mice and produce a model of the human disease.

Xenograft models of chronic phase human chronic myeloid leukemia (CML) have been difficult to develop because of the persistence of normal hematopoietic stem cells in most chronic phase CML patients and the lack of methods to selectively isolate the rarer CML stem cells. To circumvent this problem, we first identified nine patients' samples in which the long-term culture-initiating cells were predominantly leukemic and then transplanted cells from these samples into sublethally irradiated NOD/SCID and NOD/SCID-beta2microglobulin-/- mice. This resulted in the consistent and durable (>5 months) repopulation of both host genotypes with similar numbers of BCR-ABL+/Ph+ cells. The regenerated leukemic cells included an initial, transient population derived from CD34+CD38+ cells as well as more sustained populations derived from CD34+CD38- progenitors, indicative of a hierarchy of transplantable leukemic cells. Analysis of the phenotypes produced revealed a reduced output of B-lineage cells, enhanced myelopoiesis with excessive production of erythroid and megakaropoietic cells and the generation of primitive (CD34+) leukemic cells displaying an autocrine IL-3 and G-CSF phenotype, all characteristics of primary CML cells. These findings demonstrate the validity of this xenograft model of chronic phase human CML, which should enable future investigation of disease pathogenesis and new approaches to therapy.     

Stromal support augments extended long-term ex vivo expansion of hemopoietic progenitor cells.

Current technology to numerically expand hemopoietic stem/progenitor cells (HSPC) ex vivo within 1 to 2 weeks is insufficient to warrant significant gain in reconstitution time following their transplantation. In order to more stringently test the parameters affecting HSPC expansion, we followed ex vivo cultures of CD34+-selected umbilical cord blood (UCB) HSPC for up to 10 weeks and investigated the effects of stromal support and cytokine addition. The cytokine combinations included FL + TPO, FL + TPO plus SCF and/or IL6, or SCF + IL6. To identify the HSPC in uncultured and cultured material, we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ subsets by phenotyping. The highest fold-increase obtained for CD34+ and CD34+ CD38- cell numbers was, respectively, 1197 and 30,937 for stroma-free and 4066 and 117,235 for stroma-supported cultures. In general, CFC generation increased weekly in FL + TPO containing groups up to week 5 with a 28- to 195-fold expansion whereafter the weekly CFC output stabilized. Stroma support enhanced the expansion of CAFC week 6 maximally 11-fold to 89-fold with FL + TPO + IL6. Cultures stimulated with at least FL + TPO gave an estimated 10- to 14-fold expansion of the ability of CD34+ UCB cells to multilineage engraft the BM of sublethally irradiated NOD/SCID mice at 2 weeks of stroma-free and stroma-supported cultures, while at week 5 and later the estimated SRA decreased to low or undetectable levels in all groups. Our results show that stroma and FL + TPO but also inclusion of bovine serum albumin, greatly increase the long-term generation of HSPC as measured by in vitro assays and is indispensable for long-term expansion of CD34+ CD38- CXCR4+ cells. However, the different surrogate methods to quantify the HSPC (CD34+ CD38-, CFC, CAFC week 6 and SRA) show increasing incongruency with increasing culture time, while especially the phenotypic analysis and the CFC generation greatly overestimate the CAFC and SRA expansion in 10-week cultures.     

Aberrant marker expression patterns on the CD34+CD38- stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission.

Acute myeloid leukemia (AML) is generally regarded as a stem cell disease. In CD34-positive AML, the leukemic stem cell has been recognized as CD38 negative. This CD34+CD38- population survives chemotherapy and is most probable the cause of minimal residual disease (MRD). The outgrowth of MRD causes relapse and MRD can therefore serve as a prognostic marker. The key role of leukemogenic CD34+CD38- cells led us to investigate whether they can be detected under MRD conditions. Various markers were identified to be aberrantly expressed on the CD34+CD38- population in AML and high-risk MDS samples at diagnosis, including C-type lectin-like molecule-1 and several lineage markers/marker-combinations. Fluorescent in situ hybridization analysis revealed that marker-positive cells were indeed of malignant origin. The markers were neither expressed on normal CD34+CD38- cells in steady-state bone marrow (BM) nor in BM after chemotherapy. We found that these markers were indeed expressed in part of the patients on malignant CD34+CD38- cells in complete remission, indicating the presence of malignant CD34+CD38- cells. Thus, by identifying residual malignant CD34+CD38- cells after chemotherapy, MRD detection at the stem cell level turned out to be possible. This might facilitate characterization of these chemotherapy-resistant leukemogenic cells, thereby being of help to identify new targets for therapy.     

The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.

Recent studies suggest that the population of malignant cells found in human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs). LSCs have been documented for nearly all AML subtypes and have been phenotypically described as CD34+/CD38- or CD34+/HLA-DR-. Given the potentially critical role of these primitive cells in perpetuating leukemic disease, we sought to further investigate their molecular and cellular characteristics. Flow cytometric studies using primary AML tissue showed that the interleukin-3 receptor alpha chain (IL-3Ralpha or CD123) was strongly expressed in CD34+/CD38- cells (98 +/- 2% positive) from 16 of 18 primary specimens. Conversely, normal bone marrow derived CD34+/CD38- cells showed virtually no detectable expression of the CD123 antigen. To assess the functional role of IL-3Ralpha positive cells, purified CD34+/CD123+ leukemia cells were transplanted into immune deficient NOD/SCID mice. These experiments showed that CD123+ cells were competent to establish and maintain leukemic populations in vivo. To begin to elucidate a biological role for CD123 in leukemia, primary AML samples were analyzed with respect to signal transduction activity in the MAPK, Akt, and Stat5 pathways. Phosphorylation was not detected in response to IL-3 stimulation, thereby suggesting CD123 is not active in conventional IL-3-mediated signaling. Collectively, these data indicate that CD123 represents a unique marker for primitive leukemic stem cells. Given the strong expression of this receptor on LSCs, we propose that targeting of CD123 may be a promising strategy for the preferential ablation of AML cells.     

Functional cloning of IGFBP-3 from human microvascular endothelial cells reveals its novel role in promoting proliferation of primitive CD34+CD38- hematopoietic cells in vitro

Ex vivo expansion of hematopoietic stem cells (HSCs) has been investigated as a means of enhancing engraftment of transplantation therapies, but current ex vivo expansion methods typically result in a loss of functional stem cell activity. Factors that can selectively expand human HSCs remain elusive. Recently we have isolated three functionally distinct human brain microvascular endothelial cells (HBMVECs) that differ greatly in their ability to support in vitro proliferation of human umbilical cord blood (UBC) CD34+CD38-cells. Using these distinct HBMVEC populations, we have devised a cell-based functional cloning assay to identify a molecule(s) capable of facilitating expansion of HSCs in vitro. A gene encoded for IGFBP-3 (insulin-like growth factor binding protein-3) has been identified. IGFBP-3 mRNA and protein are differentially expressed in distinct HBMVEC populations. In vitro cell proliferation assay and CD34+CD38- immunophenotype analysis showed that the addition of an exogenous IGFBP-3 to cultures of purified CD34+/-CD38-Lin- cells (CD2/CD3/CD14/CD16/CD19/CD24/CD56/CD66b/GlyA depleted) enhanced proliferation of primitive hematopoietic cells with CD34+CD38- phenotype, suggesting that IGFBP-3 is capable of expanding primitive human blood cells. These expanded primitive blood cells were illustrated to maintain ability to generate functional progenitors. IGFBP-3 belongs to a family of high-affinity IGFBPs, which binds to IGFs and modulates their actions. IGFBP-3 appears to have intrinsic bioactivity that is independent of IGF binding. We are currently exploring the underlying mechanism by which IGFBP-3 modulates proliferation of primitive hematopoietic cells, and the potential of IGFBP-3 to expand pluripotent human repopulating cells capable of hematopoietic reconstitution of irradiated NOD/SCID recipients.     

In vitro proliferation and expansion of hematopoietic progenitors from patients with diffuse large B-cell lymphoma before and after chemotherapy

We have previously demonstrated that when cultured in Dexter-type Long-Term Marrow Cultures (LTMC), hematopoietic progenitor cells (HPC) from patients with Diffuse Large B-Cell Lymphoma (DLBCL) show a defective proliferation, as compared to HPC from normal marrow. In that study it was also demonstrated that functional alterations were present in the hematopoietic microenvironment developed in culture; thus, it was not clear whether such a defective proliferation in vitro was due to an intrinsic defect in the HPC compartment of DLBCL patients, or to an altered microenvironment, or both. In order to address this question, in the present study we have assessed the proliferation and expansion potentials of HPC present in bone marrow from patients with DLBCL, in cytokine-supplemented liquid cultures initiated with a cell population enriched for CD34+ Lin- cells, in the absence of stromal cells and in the presence of reduced numbers of accessory cells. Our results demonstrated that bone marrow-derived HPC from patients with DLBCL, both before and right after chemotherapy, possessed reduced proliferation and expansion potentials in vitro, as compared to their normal counterparts. Interestingly, in patients analyzed 18 months after treatment the proliferation and expansion levels were similar to those of normal HPC, indicating a complete restoration of the hematopoietic function. Although the reason for these observations is not clear, our results suggest the possibility that primitive CD34+ progenitor cells present in bone marrow, which show deficient proliferation and expansion potentials in vitro, are involved in the origin/progression of DLBCL.     

Breast cancer resistance protein in drug resistance of primitive CD34+38- cells in acute myeloid leukemia.

PURPOSE: Acute myeloid leukemia (AML) is considered a stem cell disease. Incomplete chemotherapeutic eradication of leukemic CD34+38- stem cells is likely to result in disease relapse. The purpose of this study was to investigate the role of the breast cancer resistance protein (BCRP/ATP-binding cassette, subfamily G, member 2) in drug resistance of leukemic stem cells and the effect of its modulation on stem cell eradication in AML. EXPERIMENTAL DESIGN: BCRP expression (measured flow-cytometrically using the BXP21 monoclonal antibody) and the effect of its modulation (using the novel fumitremorgin C analogue KO143) on intracellular mitoxantrone accumulation and in vitro chemosensitivity were assessed in leukemic CD34+38- cells. RESULTS: BCRP was preferentially expressed in leukemic CD34+38- cells and blockage of BCRP-mediated drug extrusion by the novel fumitremorgin C analogue KO143 resulted in increased intracellular mitoxantrone accumulation in these cells in the majority of patients. This increase, however, was much lower than in the mitoxantrone-resistant breast cancer cell line MCF7-MR and significant drug extrusion occurred in the presence of BCRP blockage due to the presence of additional drug transport mechanisms, among which ABCB1 and multiple drug resistance protein. In line with these findings, selective blockage of BCRP by KO143 did not enhance in vitro chemosensitivity of leukemic CD34+38- cells. CONCLUSIONS: These results show that drug extrusion from leukemic stem cells is mediated by the promiscuous action of BCRP and additional transporters. Broad-spectrum inhibition, rather than modulation of single mechanisms, is therefore likely to be required to circumvent drug resistance and eradicate leukemic stem cells in AML.     

The immunophenotype of minimally differentiated acute myeloid leukemia (AML-M0): reduced immunogenicity and high frequency of CD34+/CD38- leukemic progenitors.

Minimally differentiated acute myeloid leukemia (AML-M0) is a rare FAB subtype (2-3% of AMLs) of poor prognosis. The aim of our study was to characterize AML-M0 expression and regulation of adhesion/costimulatory molecule involved in immune recognition, to test blast in vitro immunogenicity, and to determine the percentage of leukemia progenitor cells. Here, we demonstrate that alloimmune recognition of AML-M0 in primary mixed lymphocyte reaction, as evaluated by IL-2 secretion of responding T cells, is reduced in comparison with more differentiated subtypes (128 +/- 95 pg/ml vs304 +/- 159 pg/ml, P < 0.05). These data are in line with low blast cell expression of major histocompatibility complex (MHC) class II DR molecules, and of the CD28 ligand B7-2, which plays an important role in AML immune recognition. Adhesion/costimulatory molecules were up-regulated by leukemic cell stimulation via CD40, and, although less efficiently, by gamma-IFN; both stimuli improved blast cell immunogenicity. We also demonstrate that AML-M0 have a very high percentage (40% +/- 30) of CD34+/CD38- leukemic clonogenic precursors in comparison with more differentiated AMLs (2.5% +/- 2) or non-leukemic CD34+hematopoietic precursors (1.8% +/- 0.8). Since the presence of a leukemic cell population at an early differentiation stage has been identified as a poor prognostic factor, we conclude that the high frequency of CD34+/CD38- blasts in AML-M0 may converge with already identified poor prognosis factors such as chemotherapy resistance and cytogenetic abnormalities. The clinical implications of AML-M0 impaired in vitroimmunogenicity and a high percentage of CD34+/CD38- blasts will require comparative analysis of additional patients. The increased immunogenicity of blast cells after CD40 triggering provide interesting clues for AML-M0 immunotherapy, that have to be confirmed with an in vivo leukemia model in mice.     

Sequential analysis of CD34+ and CD34- cell subsets in peripheral blood and leukapheresis products from breast cancer patients mobilized with SCF plus G-CSF and cyclophosphamide.

Administration of stem cell factor (SCF) has been proven to enhance cytokine-induced mobilization of CD34+ hematopoietic progenitor cells (HPC) into the peripheral blood (PB). The aim of the present study was to explore in a homogeneous group of 22 uniformly treated breast cancer patients: (1) the kinetics of mobilization into PB of both CD34+ and CD34- cell subsets, including dendritic cells, in sequential samples obtained from day +7 up to day +12 after mobilization; and (2) the composition of the CD34+ and CD34- cell subsets present in the two leukapheresis products obtained for each patient. The following CD34+ and CD34- subsets were analyzed: early CD34+ HPC, erythroid-, myeloid- and B-lymphoid-committed CD34+ precursor cells, mature T, B and NK cells, monocytes, neutrophils, eosinophils, basophils, and dendritic cells (DC) including three subsets of lin-/HLADR+DC (CD16+, CD33high and CD123high). Our results show that the absolute number of PB CD34+ HPC progressively increases from day +7 onwards. As far as the CD34- PB leukocyte subsets are concerned, monocytes (CD14+) displayed the earliest recovery after mobilization predicting neutrophil recovery 1 day in advance. The number of CD34+ HPC collected in a single leukapheresis product was always > or = 1.4 x 10(6) cells/kg body weight. No significant changes were observed between the two leukapheresis sessions either as regards their composition in CD34+ HPC subsets or their CD34- leukocyte populations except for a higher ratio of both CD34+ erythroid/CD34+ myeloid HPC (0.35 +/- 0.13 vs 0.30 +/- 0.13; P = 0.04) and neutrophils/monocytes (1.58 +/- 2.1 vs 0.69 +/- 0.27; P = 0.009) found for the first leukapheresis. Interestingly, the overall number of dendritic cells (DC) was higher in the second leukapheresis (1.06 +/- 0.56 vs 1.9 +/- 0.46; P = 0.02) due to a selective increase of the CD16+ antigen-presenting cells. In summary, our results show that the combination of cyclophosphamide, G-CSF and SCF is highly effective for stem cell mobilization, with differences observed in the mobilization kinetics of the different hematopoietic cell subsets analyzed.     

Functional alterations of Lin-CD34+CD38+ cells in chronic myelomonocytic leukemia and on progression to acute leukemia

The functional behavior of hematopoietic stem cell (HSC) and progenitors in chronic myelomonocytic leukemia (CMML) and on disease progression is little known. We performed cell proliferation, apoptosis, hematopoietic colony forming/replating and differentiation potential studies in the purified subpopulations of Lin(-)CD34(+)CD38(-) and Lin(-)CD34(+)CD38(+) cells from 16 CMML with 6 cases after acute myeloid leukemia transformation (AML-t). We observed an expansion of the hematopoietic progenitor pool (Lin(-)CD34(+) cells) in AML-t comprising mainly Lin(-)CD34(+)CD38(+) cells. The Lin(-)CD34(+)CD38(+) cells in AML-t displayed high proliferative activity, resistance to apoptosis, enhanced myeloid colony formation/replating ability and a complete dendritic cell (DC) differentiation block. Our findings suggest Lin(-)CD34(+)CD38(+) cells instead of Lin(-)CD34(+)CD38(-) cells could be the target(s) of secondary genetic lesions underpinning progression from CMML to AML, which have implications for the further study of the biology of leukemic transformation and the design of new strategies for the effective treatment of CMML.